VEGF receptor expression decreases during lung development in congenital diaphragmatic hernia induced by nitrofen

Changes in vascular endothelial growth factor (VEGF) in pulmonary vessels have been described in congenital diaphragmatic hernia (CDH) and may contribute to the development of pulmonary hypoplasia and hypertension; however, how the expression of VEGF receptors changes during fetal lung development in CDH is not understood. The aim of this study was to compare morphological evolution with expression of VEGF receptors, VEGFR1 (Flt-1) and VEGFR2 (Flk-1), in pseudoglandular, canalicular, and saccular stages of lung development in normal rat fetuses and in fetuses with CDH. Pregnant rats were divided into four groups (n=20 fetuses each) of four different gestational days (GD) 18.5, 19.5, 20.5, 21.5: external control (EC), exposed to olive oil (OO), exposed to 100 mg nitrofen, by gavage, without CDH (N-), and exposed to nitrofen with CDH (CDH) on GD 9.5 (term=22 days). The morphological variables studied were: body weight (BW), total lung weight (TLW), left lung weight, TLW/BW ratio, total lung volume, and left lung volume. The histometric variables studied were: left lung parenchymal area density and left lung parenchymal volume. VEGFR1 and VEGFR2 expression were determined by Western blotting. The data were analyzed using analysis of variance with the Tukey-Kramer post hoc test. CDH frequency was 37% (80/216). All the morphological and histometric variables were reduced in the N- and CDH groups compared with the controls, and reductions were more pronounced in the CDH group (P<0.05) and more evident on GD 20.5 and GD 21.5. Similar results were observed for VEGFR1 and VEGFR2 expression. We conclude that N- and CDH fetuses showed primary pulmonary hypoplasia, with a decrease in VEGFR1 and VEGFR2 expression.

Vitamin A Effect to Nitrofen Induced Congenital Diaphragmatic Hernia and Pulmonary Hypoplasia in Rats

PURPOSE: Vitamin A has been introduced recently for its feasible effect in curing diaphragmatic defect and accelerating lung development during the perinatal period of experimental rats or humans suffering from congenital diaphragmatic hernia (CDH). Despite continual research attention since the fifties to elucidate the influence and mechanisms of vitamin A on pulmonary growth, many presumptive hypotheses remain, along with an inherently high mortality. So we wondered whether prenatal vitamin A alone or combined with dexamethasone could accomplish better results than dexamethasone against the diaphragmatic defect or lung hypoplasia in neonatal rats. METHODS: Pregnant Sprague-Dawley rats exposed to Nitrofen were classified into 5 groups according to the different treatment options. Studies were performd in 2 phases. In study 1, the 24-hour survival rate and preliminary results were observed. In study 2, the incidence and site of CDH, lung/body weight ratio (L/BWR), radial saccular counts (RSC) and maturation of alveolar sac in 3 histomorphologic grades were evaluated among the 5 groups. RESULTS: Vitamin A treated neonatal rats (group III) showed improved lung development compared with rats without treatments (group II) in 24-hour survival rate, L/BWR and alveolar maturation (P<0.001), leading to lung development that was comparable in every aspect to that of the dexamethasone treated rats (group IV). Combined treatment by vitamin A and dexamethasone (group V) improved the incidence of CDH, L/BWR (P<0.001), RSC (P<0.05) and alveolar maturation (P<0.001) when compared with rats treated alone by vitamin A (group III) or dexamethasone (group IV), leading to a level of development that was closest to that of the normal control lungs (group I). CONCLUSION: Vitamin A had a therapeutic effect on pulmonary hypoplasia in the experimental rats, and when combined with dexamethasone it accomplished a better outcome in the treatment of CDH itself or pulmonary hypoplasia. After the problem of vitamin A toxicity is settled, the future of vitamin A as a prenatal therapeutic agent for CDH might gain in appeal.

Congenital Diaphragmatic Hernia in Rats Using Nitrofen: An Animal Model

PURPOSE: Since 1971, Nitrofen (2,4-dichloro-4`-nitrodiphenyl ether) herbicide has been known to induce variable congenital abnormalities in rats. However, until now there has been no animal model of congenital diaphragmatic hernia (CDH) using this herbicide in Korea. Therefore, CDH in rats using Nitrofen was tried for searching on pathogenesis or using therapeutic modalities of CDH. METHODS: 20 Sprague-Dawley rats ingested Nitrofen as indicated on the 9.5th day of gestation, and after sacrificing on the 21th day of gestation. 243 offspring were harvested for the examination of diaphragmatic herniations. Dissections were made along sites of herniations to confirm the diaphragmatic herniation's site and size, lung/body weight ratio comparison as well as to accomplish microscopic radial saccular counting and evaluation of alveolar septal wall maturations and a comparision was made between the groups. RESULTS: CDH appeared in 149 of 243 offspring. CDH only appeared in the posterior portion of the diaphragm regardless of size or the side of appearance. Left sided CDH was the most common single diaphragmatic anomaly, represented in 112 offspring. Right sided CDH was next, found in 31 offspring, and bilateral CDH was seen in 6 offspring. Left sided CDH was almost always associated with visceral herniations and with pulmonary hypoplasia on the affected side, which was proved in the decreased Lung/Body weight ratio and in decreased radial saccular counts and in increased alveolar septal wall thickness in the affected lungs of CDH. In94 offspring without CDH following exposure to Nitrofen, moderate pulmonary hypo plasia was microscopically observed. CONCLUSION: Initial Rat modeling of congenital diaphragmatic hernia using Nitrofen showed results of herniation and pulmonary hypoplasia of the affected lungs acceptable for further experimental studies on CDH and accompaning pulmonary abnormalities.